Intracellular calcium (Ca2+) signaling events that differ in respect to spatial localization, temporal kinetics, and physiological function occur in a wide variety of cell types. In arterial smooth muscle cells, three different types of intracellular Ca2+ signaling modalities have been described; localized transients termed "Ca2+ sparks," propagating events termed "Ca2+ waves," and global intracellular Ca2+ concentration ([Ca2+]i) elevations. Preliminary data from our laboratory suggest that intravascular pressure elevates Ca2+ spark frequency, Ca2+ wave frequency and global [Ca2+]I in cerebral artery smooth muscle cells by inducing a steady membrane depolarization that activates voltage-dependent Ca2+ channels. Our data also suggest that pressure induces constriction ("myogenic tone") via an elevation of global [Ca2+] whereas sparks and waves, which occur due to the activation of ryanodine-sensitive Ca2+ release (RyR) channels on the sarcoplasmic reticulum (SR), do not contribute significantly to global [Ca2+]I, and the net effect of sparks and waves is to oppose constriction. In this proposal we will test the hypothesis that intravascular pressure activates different intracellular Ca2+ signaling modalities in cerebral artery smooth muscle cells via activation of voltage-dependent Ca2+ channels and investigate mechanisms of signaling between voltage-dependent Ca2+ channels and RyR channels. We will employ several state-of-the-art techniques including laser scanning confocal Ca2+ imaging, ratiometric Ca2+ imaging, patch clamp electrophysiology, and diameter measurements of pressurized arteries. We propose 3 Specific Aims. Aim 1 will investigate the regulation of intracellular Ca2+ signaling modalities in cerebral artery smooth muscle cells and arterial diameter by intravascular pressure, and explore the hypothesis that pressure activates Ca2+-dependent potassium (BKca) channels by inducing intracellular Ca2+ release events. Aim 2 will examine the hypothesis that steady membrane depolarization activates Ca2+ sparks via an elevation of cytosolic [Ca2]i and SR Ca2+ load. Aim 3 will investigate the hypothesis that localized subsarcolemmal [Ca2+]I elevations caused by the opening of voltage-dependent Ca2+ channels activate Ca2+ sparks in cerebral artery smooth muscle cells. This work will provide a better understanding of the regulation and physiological functions of Ca2+ signaling modalities in cerebral artery smooth muscle cells.